Cardiovascular disease (CVD) represents the major source of morbidity and mortality. Despite maximal therapeutic intervention, significant risks remain. There is a critical need for discovery of pathways linked to CVD pathogenesis. The proposed studies combine unbiased metabolomics approaches, with more quantitative targeted analyses, and mechanistic animal models and human studies, to define novel analytes and pathways linked to CVD pathogenesis. In aim 1, we propose new mechanistic studies in animal models of disease, and in humans, centered around a new and unpublished pathway discovered recently during performance of our initial unbiased metabolomics studies. We have accrued substantial preliminary data in both mouse models and human clinical investigations showing a dietary nutrient abundant in red meat is metabolized by gut flora and is linked to atherosclerosis. Unpublished targeted metabolomics studies, isotope tracer studies in germ free mice and humans antibiotic suppression, and additional mechanistic animal model studies, collectively indicate at least three distinct metabolites produced by gut flora may possess direct biological activity impacting upon atherosclerosis susceptibility and cholesterol metabolism. In aim 2 we propose to substantially extend upon our initial unbiased metabolomics investigations using two distinct and well defined clinical cohorts. Studies in subjects undergoing elective cardiac evaluations and for whom long term follow-up is available will enable us to discover new analytes and pathways associated with development of atherosclerotic heart disease and its adverse complications heart attack, heart failure and death. While metabolomics studies in another, a clinical trial monitored by sequential coronary intravascular ultrasound [IVUS]), will allow us to define analytes and pathways associated with response to therapy for high potency statins. Successful completion of our proposed research program will provide opportunities for development of both novel diagnostic tests and therapeutic approaches for the treatment and prevention of atherosclerotic heart disease and its major complications. It will also further establish gut flora-dependent metabolism of an abundant nutrient in red meat as a new pathway for cardiovascular disease pathogenesis discovered through unbiased metabolomics.